Imine reductase mutant and application thereof in synthesis of 2-methyl-10-ethyl denitriflavin

Abstract

The invention relates to an imine reductase mutant and an application of the imine reductase mutant in synthesis of 2-methyl-10-ethyl denitroflavin. The amino acid sequence of the imine reductase mutant is as shown in SEQ ID NO. 2, and the nucleotide sequence of the imine reductase mutant is as shown in SEQ ID NO. 1. The invention also provides a method for synthesizing 2-methyl-10-ethyl denitriflavin by using the imine reductase mutant. The method comprises the following steps: (1) preparing supernate containing the imine reductase mutant by a heterologous expression method; (2) respectively preparing a solution A and a solution B by taking (5Z)-5-[(2-ethylaminophenyl) methylene]-1-methylpyrimidine-2, 4, 6 (1H, 3H, 5H)-triketone, D-glucose, glutamate dehydrogenase and reduced nicotinamide adenine dinucleoside phosphate as raw materials; and (3) carrying out reaction on the solution A and the solution B to prepare the 2-methyl-10-ethyl denitroflavin. The imine reductase mutant and the synthesis method provided by the invention overcome the defects of complex route, high cost, difficult purification, serious environmental pollution and the like of a chemical preparation method, and provide a green way for synthesis of 2-methyl-10-ethyl denitriflavin.

Description

TECHNICAL FIELD

[0001] The application relates to an imine reductase mutant and application thereof in synthesis of 2-methyl-10-ethyl deazaflavin, and belongs to the technical field of genetic engineering. BACKGROUND

[0002] With the increase of age, the content of collagen in the skin gradually decreases, and the structure and function of collagen fibers also change, resulting in loss of skin elasticity and appearance of wrinkles. Therefore, supplementing collagen and promoting synthesis of collagen are important ways for anti-wrinkles.

[0003] Deazaflavin compounds are an important class of bioactive molecules, which participate in various biological oxidation-reduction reactions as coenzyme analogs and have wide applications in the fields of medicine, agriculture and biological catalysis. As a derivative of deazaflavin, 2-methyl-10-ethyl deazaflavin has high biological activity, can promote the in-vivo production of NAD + , can activate sirtuins, helps to delay aging, can repair mitochondrial membrane potential and improve ATP generation efficiency; has good stability and can maintain its activity and effect under different environments and storage conditions; has stronger antioxidant properties and can neutralize free radicals generated during mitochondrial electricity generation to create a stable internal environment for mitochondria; can directly act as a coenzyme and can directly insert into the "energy lock hole" of mitochondria to activate the energy metabolism-related links.

[0004] Traditional methods for synthesizing deazaflavin compounds mostly rely on pure chemical synthesis, which has problems such as complicated reaction steps, poor stereoselectivity, use of toxic reagents (such as heavy metal reducing agents) and low environmental friendliness. In recent years, biological catalysis technology has gradually been applied to the synthesis of complex compounds due to its advantages such as mild conditions, high selectivity and environmental friendliness. Aspergillus niger is a widely existing filamentous fungus, and its genome encodes a variety of imine reductases with potential catalytic activity. The enzyme can catalyze the reductive coupling reaction of a variety of carbonyl compounds and amines. When both the carbonyl compound and the amine have high reactivity, a 1:1 substrate ratio can be used for the reaction, which provides a green route for the synthesis of nitrogen-containing heterocyclic compounds.

[0005] However, there is no report on the enzymatic synthesis of 2-methyl-10-ethyl deazaflavin, so it is of great significance to develop an imine reductase that can synthesize 2-methyl-10-ethyl deazaflavin and a high-efficiency synthesis route combining chemical method and enzymatic method. SUMMARY

[0006] In view of the deficiencies of the prior art, an imine reductase mutant and application thereof in synthesis of 2-methyl-10-ethyl deazaflavin are provided.

[0007] The technical scheme of the present application is as follows:

[0008] An imine reductase mutant, the amino acid sequence of which is shown as SEQ ID NO. 2, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

[0009] The imine reductase mutant provided by the present application is based on the imine reductase gene derived from Aspergillus niger, in which the valine (Val) at the 106th position is mutated to aspartic acid (Asp), the methionine (Met) at the 214th position is mutated to isoleucine (Ile), the aspartic acid (Asp) at the 226th position is mutated to tyrosine (Tyr), the glutamine (Gln) at the 233rd position is mutated to proline (Pro), and the alanine (Ala) at the 266th position is mutated to glutamic acid (Glu); the GenBank accession number of the imine reductase is KY327363.1.

[0010] A recombinant vector is obtained by inserting the coding gene of the imine reductase mutant into a plasmid vector.

[0011] According to the present application, the plasmid vector is preferably pET-28a(+).

[0012] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host cell.

[0013] According to the present application, the host cell is preferably Escherichia coli.

[0014] The imine reductase mutant is used in the synthesis of 2-methyl-10-ethyl deazaflavin.

[0015] A method for synthesizing 2-methyl-10-ethyl deazaflavin, comprising the following steps:

[0016] (1) The imine reductase mutant gene is cloned into an expression vector to obtain a recombinant expression vector; then the recombinant expression vector is transformed into a host cell, and a positive clone is selected for fermentation culture, and wet bacteria are collected; then the obtained wet bacteria are added to PBS buffer, and ultrasonic crushing is performed for 35-45 min, followed by centrifugation at 8000-12000 rpm for 5-15 min to obtain a supernatant containing the imine reductase mutant;

[0017] (2) (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione is dissolved in dimethyl sulfoxide (DMSO) to form solution A; D-glucose, glutamate dehydrogenase (GDH), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are added into PBS buffer to form solution B;

[0018] (3) Solution A and the supernatant containing the imine reductase mutant obtained in step (1) are added into solution B, and stirred to mix uniformly, and then reacted at 35-40 °C and 200-300 rpm for 20-30 h, and then the reaction is terminated by adding NaOH, and after extraction and drying, 2-methyl-10-ethyl deazaflavin is obtained.

[0019] According to the application, in step (1), the PBS buffer has a pH of 7.5, and the mass-volume ratio of the wet bacteria to the buffer is 10:1, unit: mL / g; the ultrasonic cell disrupter is used to treat the bacteria at a power of 150-300 kw for 40 min, with a working time of 3 s and a stopping time of 3 s.

[0020] According to the application, in step (2), the (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione has the following formula (1):

[0021]

[0022] The (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione is prepared according to the following method:

[0023] 1-methylbarbituric acid and 2-ethylaminobenzaldehyde are added into a reaction container containing ethanol to form a reaction system; the reaction system is stirred and reacted at 60-90 °C for 4-8 h, and after the reaction is completed, the reaction solution is cooled to room temperature, and solid is precipitated, which is filtered and recrystallized with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione.

[0024] The mass ratio of 1-methylbarbituric acid to 2-ethylaminobenzaldehyde is 1:(1-1.2); and the volume-mass ratio of ethanol to 1-methylbarbituric acid is (10-20):1, unit: mL / g.

[0025] According to the application, in step (2), the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in solution A is 0.2-0.3 g / mL.

[0026] According to the application, preferably, in step (2), the concentration of D-glucose in solution B is 55-65 mM, the concentration of glutamate dehydrogenase (GDH) is 55-65 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 35-45 mM.

[0027] According to the application, preferably, in step (3), the volume ratio of solution A, solution B and supernatant containing the imine reductase mutant is (45-55):(440-460):10.

[0028] According to the application, preferably, in step (3), the termination reaction, extraction and drying are specifically as follows:

[0029] First, the reaction is terminated by adding 5M NaOH, then the reaction solution is extracted twice with 3 times the volume of ethyl acetate, the organic phases obtained by extraction are combined, and finally the combined organic phase is dried with anhydrous MgSO4.

[0030] The technical features and advantages of the application are as follows:

[0031] 1. The application is based on the imine reductase (GenBank accession number: KY327363) derived from Aspergillus niger, which is mutated to obtain an imine reductase mutant in which valine (Val) at position 106 is mutated to aspartic acid (Asp), methionine (Met) at position 214 is mutated to isoleucine (Ile), aspartic acid (Asp) at position 226 is mutated to tyrosine (Tyr), glutamine (Gln) at position 233 is mutated to proline (Pro), and alanine (Ala) at position 266 is mutated to glutamic acid (Glu), and the mutant is applied in the synthesis of 2-methyl-10-ethyl deazaflavin.

[0032] 2. Compared with the wild-type imine reductase, the enzyme activity of the imine reductase mutant provided by the application is significantly improved, which can more effectively catalyze the reductive coupling reaction of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione and D-glucose, improve the utilization rate of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione, and greatly improve the synthesis efficiency and purity of 2-methyl-10-ethyl deazaflavin.

[0033] 3. The method for synthesizing 2-methyl-10-ethyl deazaflavin provided by the application overcomes the shortcomings of chemical preparation methods, such as complex route, high cost, difficult purification, and serious environmental pollution, and provides a green route for the synthesis of 2-methyl-10-ethyl deazaflavin. Moreover, the reaction steps are short and the conditions are mild, which significantly reduces equipment investment and energy consumption cost, and is suitable for industrialized production. Attached Figure Description

[0034] Figure 1 Agarose gel electrophoresis images of imine reductase mutant V106D-M214I-D226Y-Q233P-A266E and wild-type imine reductase;

[0035] In the figure, lane 1 represents wild-type imine reductase, and lane 2 represents imine reductase mutant.

[0036] Figure 2 This is a liquid phase diagram of 2-methyl-10-ethyl desoxyflavin prepared in Example 4.

[0037] Figure 3 The liquid phase diagram is for 2-methyl-10-ethyl desoxyflavin prepared in Comparative Example 1. Detailed Implementation

[0038] The following embodiments and accompanying drawings are merely illustrative of specific implementation schemes for carrying out the present invention. These schemes and drawings should not be construed as limiting the present invention. Any changes made without departing from the principles and essence of the present invention shall fall within the protection scope of the present invention.

[0039] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional. Unless otherwise specified, all materials and reagents used in this embodiment can be obtained through legitimate commercial channels.

[0040] To better explain this invention, the following embodiments further illustrate the invention. Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific molecular biology experimental methods not specifically described, please refer to *Molecular Cloning: A Laboratory Manual* edited by J. Sambrook et al., or follow the kit instructions. Unless otherwise specified, the reagents and biological materials used in the specific embodiments are commercially available.

[0041] Example 1: Selection of mutation sites

[0042] Homology modeling was performed on the amino acid sequence shown in the wild-type imine reductase nucleotide sequence (GenBank accession number: KY327363.1), and then docked with the substrate molecule. The results were then imported into Discovery Studio software for visualization analysis. The amino acids that interact with the substrate were analyzed, and V106, M214, D226, Q233, and A266 were selected as potential mutation sites.

[0043] Example 2: Preparation of imine reductase mutant

[0044] 1. The nucleotide sequence of wild-type imine reductase (GenBank Accession No. KY327363.1) was artificially synthesized by Jui Zhi Biotechnology Co., Ltd., and then the coding gene sequence was cloned into pET-28a plasmid to obtain the recombinant plasmid pET-28a-AspRedAm expressing wild-type imine reductase AspRedAm. The recombinant plasmid was transformed into competent cells of Escherichia coli BL21 (DE3) by calcium chloride method to obtain the recombinant E. coli expressing imine reductase AspRedAm

[0045] BL21-pET-28a-AspRedAm, stored at -80℃.

[0046] 2. The AspRedAm mutant gene was obtained through five rounds of iterative saturation mutation and preliminary screening of dominant strains, and the specific method was as follows:

[0047] The first round: using the recombinant plasmid pET-28a-AspRedAm as a template and F106 and R106 as primers, the isoleucine at position 106 of the amino acid sequence was mutated to the remaining 19 amino acids by site-directed saturation mutation PCR, and then E. coli was transformed, spread on LB solid medium and cultured, and through dominant strain screening, the recombinant E. coli BL21-pET-28a-AspRedAm-V106D was obtained.

[0048] BL21-pET-28a-AspRedAm-V106D and other 18 kinds of recombinant E. coli;

[0049] The screening method of dominant strains: single colonies of mutants were selected from LB solid medium, inoculated into 96-well plates containing 500 μL of LB liquid medium, and cultured at 37℃, 220 rpm for 12 h. According to the inoculation amount of 2% (v / v), it was inoculated into 96-well plates containing 500 μL of LB liquid medium, and cultured at 37℃, 220 rpm for 3 h. After adding IPTG with a final concentration of 0.1 mmol / L, it was induced and cultured at 20℃ for 12 h to obtain the fermentation broth. The fermentation broth was centrifuged to obtain wet bacteria, and the wet bacteria were ultrasonically broken to obtain the broken supernatant containing imine reductase mutants.

[0050] 14 mg of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione was dissolved in 50 μL of DMSO to obtain solution A;

[0051] Take 450 μL of phosphate buffer with pH = 7.5, and add 60 mM D-glucose, 60 mg / mL GDH, 40 mM NADPH and 10 μL of the broken supernatant containing imine reductase mutants in sequence, mix well and dissolve completely to obtain solution B. Mix solution A and B, and place in a 37°C, 250 rpm shaker for 24 h. Then add 5M NaOH to terminate the reaction, extract twice with ethyl acetate, combine the organic phases, and take an appropriate amount into HPLC for detection and analysis. The strain with high 2-methyl-10-ethyl deazaflavin content is the dominant strain, and the screening result is recombinant Escherichia coli BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P.

[0052] BL21-pET-28a-AspRedAm-V106D.

[0053] The second round: taking the recombinant plasmid extracted from the strain BL21-pET-28a-AspRedAm-V106D as a template, and taking F214 and R214 as primers, the methionine at position 214 of the amino acid sequence is mutated to the remaining 19 kinds of amino acids by site-directed saturation mutation PCR, and then the Escherichia coli is transformed, coated with LB solid medium and cultured, and the dominant strain is screened according to the method described in the first round. The recombinant Escherichia coli BL21-pET-28a-AspRedAm-V106D-M214I is obtained.

[0054] The third round: taking the recombinant plasmid extracted from the strain BL21-pET-28a-AspRedAm-V106D-M214I as a template, and taking F226 and R226 as primers, the aspartic acid at position 226 of the amino acid sequence is mutated to the remaining 19 kinds of amino acids by saturation mutation PCR, and then the Escherichia coli is transformed, coated with LB solid medium and cultured, and the dominant strain is screened according to the method described in the first round. The recombinant Escherichia coli BL21-pET-28a-AspRedAm-V106D-M214I-D226Y is obtained.

[0055] The fourth round: taking the recombinant plasmid extracted from the strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y as a template, and taking F233 and R233 as primers, the glutamine at position 233 of the amino acid sequence is mutated to the remaining 19 kinds of amino acids by site-directed saturation mutation PCR, and then the Escherichia coli is transformed, coated with LB solid medium and cultured, and the dominant strain is screened according to the method described in the first round. The recombinant Escherichia coli

[0056] BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P.

[0057] The 5th round: taking the recombinant plasmid extracted from the strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P as a template, taking F266 and R266 as primers, mutating the amino acid sequence of 266th position from alanine to the remaining 19 kinds of amino acids by saturated mutation PCR, then transforming E. coli, coating LB solid medium and culturing, screening dominant strains by the method described in the 1st round, obtaining recombinant E. coli

[0058] BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266E.

[0059] The system (25 μL) of the site-directed saturated mutation PCR is shown in Table 1.

[0060] Table 1

[0061] Sample Amount added (μL) Final concentration (mM) 10 x PCR 2.5 / dGTP (10 mM) 0.5 0.2 dATP (10 mM) 0.5 0.2 dCTP (10 mM) 2.5 1.0 dTTP (10 mM) 2.5 1.0 Primer-F (10 mM) 0.5 0.2 Primer-R (10 mM) 0.5 0.2 Mn 2+ (20mM)]]> 0.375 0.3 Mg 2+ (25mM)]]> 7 7 Template (16-18 ng / μL) 3 2 ng / μL Taq enzyme (5 U / μL) 1 0.1 Water (μL) 4.125 / Final volume (μL) 25 /

[0062] PCR conditions: 95°C pre-denaturation for 5 min, 30 cycles of 90°C for 30 s, 62°C for 30 s, 72°C for 7 min, and finally 72°C for 5 min.

[0063] The primers of the site-directed saturated mutation PCR are shown in Table 2.

[0064] Table 2

[0065] Primer name Primer sequence (5'-3') F106 GGTGCAATGGGTACCGCACTGNNKTGCGAAATACCTGGAGCAT R106 ATGCTCCAGGTATTTGCGACMNNAGTGCGGTACCCATTGCACC F214 GGTATCATGGCGGTGCCGACCNNKGATTGGCTCTCCGCACG R214 CGTGCGGAGAGCCAATCMNNGGTCGGCACC GCCATGATACC F226 GGTGAATCCCTGGAACTGTTTNNKATTGAATCTCACCTGTCT R226 AGACAGGTGAGATTCAATMNNAAACAGTTCCAGGGATTCACCG F233 CTGATTAAATCTGGTCAGGACNNKACCACTGCAACTGGTCTG R233 CAGACCAGTTGCAGTGGTMNNGTCCTGACCAGATTT AATCAG F266 GAAGAACAGCGTGTTTCTTCTNNKATGATCCTGCCGATCAAAG R266 CTTTGATCGGCAGGATCATMNNGAAGAAACACGCTGTTCTTC

[0066] The following 13 recombinant E. coli strains were constructed and preliminarily screened according to the above method, and were respectively recombinant E. coli BL21-pET-28a-AspRedAm-V106D, BL21-pET-28a-AspRedAm-V106E, BL21-pET-28a-AspRedAm-V106D-M214I, BL21-pET-28a-AspRedAm-V106D-M214L, BL21-pET-28a-AspRedAm-V106D-M214V, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y, BL21-pET-28a-AspRedAm-V106D-M214I-D226F, BL21-pET-28a-AspRedAm-V106D-M214I-D226W, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233G, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266E, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266D, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266Q.

[0067] 3. Secondary screening of enzyme mutants

[0068] (1) Preparation of enzyme mutants: taking the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E as an example, the strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266E was inoculated into LB liquid medium containing 50 mg / L kanamycin, and cultured for 12 h, then inoculated into fresh LB liquid medium containing 50 mg / L kanamycin at a 2% (v / v) inoculation amount, and cultured until the OD600 value was 0.6-0.8, then 0.1 mmol / L IPTG was added, and the culture was induced at 20°C for 12 h to obtain a fermentation broth; the fermentation broth was centrifuged to obtain wet bacteria, and the wet bacteria were ultrasonically broken in phosphate buffer, and then centrifuged to obtain the supernatant, which was the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E.

[0069] V106D-M214I-D226Y-Q233P-A266E.

[0070] Wild-type imine reductase, imine reductase mutant V106D, imine reductase mutant V106E, imine reductase mutant V106D-M214I, imine reductase mutant V106D-M214L, imine reductase mutant V106D-M214V, imine reductase mutant V106D-M214I-D226Y, imine reductase mutant

[0071] V106D-M214I-D226F, imine reductase mutant V106D-M214I-D226W, imine reductase mutant

[0072] V106D-M214I-D226Y-Q233P, imine reductase mutant V106D-M214I-D226Y-Q233G, imine reductase mutant V106D-M214I-D226Y-Q233P-A266E, imine reductase mutant

[0073] V106D-M214I-D226Y-Q233P-A266D, imine reductase mutant V106D-M214I-D226Y-Q233P-A266Q and wild-type imine reductase AspRedAm.

[0074] The imine reductase mutant V106D-M214I-D226Y-Q233P-A266E and wild-type imine reductase were subjected to DNA agarose gel electrophoresis verification, and the results are shown in Figure 1

[0075] It can be known from Figure 1 that a target band appears in the gel electrophoresis diagram and the band is single, which indicates that the imine reductase gene mutation is successful, and the imine reductase mutant

[0076] V106D-M214I-D226Y-Q233P-A266E is successfully prepared by heterologous expression.

[0077] ​(2) The catalytic efficiency of wild-type imine reductase, imine reductase mutant V106D, imine reductase mutant V106E, imine reductase mutant V106D-M214I, imine reductase mutant V106D-M214L, imine reductase mutant V106D-M214V, imine reductase mutant V106D-M214I-D226Y, imine reductase mutant V106D-M214I-D226F, imine reductase mutant V106D-M214I-D226W, imine reductase mutant V106D-M214I-D226Y-Q233P, imine reductase mutant V106D-M214I-D226Y-Q233G, imine reductase mutant V106D-M214I-D226Y-Q233P-A266E, imine reductase mutant V106D-M214I-D226Y-Q233P-A266Q on the synthesis reaction of 3-methyl-10-ethyl deazaflavin was determined according to the method described in item 2, and the results are shown in Table 3.

[0078] V106D-M214I-D226Y-Q233P-A266D, imine reductase mutant V106D-M214I-D226Y-Q233P-A266Q on the synthesis reaction of 3-methyl-10-ethyl deazaflavin was determined according to the method described in item 2, and the results are shown in Table 3.

[0079] Table 3

[0080]

[0081]

[0082] As can be seen from Table 3, the enzyme activities of the imine reductase mutants are all higher than that of the wild-type imine reductase, and the catalytic efficiency of imine reductase mutant V106D-M214I-D226Y-Q233P-A266E on the synthesis reaction of 3-methyl-10-ethyl deazaflavin is the best, with a content of 98.5%, which is more than 20% higher than that of the wild-type imine reductase.

[0083] Example 3, Preparation of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione

[0084] A method for preparing (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione, the steps are as follows:

[0085] A reaction system was constructed by adding 15 g of 1-methylbarbituric acid and 15 g of 2-ethylaminobenzaldehyde into a reaction vessel containing 225 mL of ethanol; the reaction system was stirred at 75°C for 5 h, after the reaction was completed, the reaction solution was cooled to room temperature, and a solid was precipitated, which was filtered and recrystallized with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione.

[0086] Example 4, synthesis of 2-methyl-10-ethyl deazaflavine

[0087] A method for synthesizing 2-methyl-10-ethyl deazaflavine, comprising the following steps:

[0088] (1) 14 g of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione was dissolved in 50 mL of dimethyl sulfoxide (DMSO) to obtain solution A; D-glucose, glutamate dehydrogenase (GDH), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were added to 450 mL of PBS buffer to obtain solution B;

[0089] In solution A, the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione was 0.28 g / mL; in solution B, the concentration of D-glucose was 60 mM, the concentration of glutamate dehydrogenase (GDH) was 60 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) was 40 mM;

[0090] (2) 450 mL of solution A and 10 mL of supernatant containing imine reductase mutant obtained in Example 3 were added to 50 mL of solution B, and stirred to mix uniformly, and then reacted at 37°C and 250 rpm for 24 h, and then terminated by adding 5M NaOH, and then extracted twice with 3 times the volume of ethyl acetate, and then the organic phases obtained by extraction were combined, and finally dried with anhydrous MgSO to obtain 2-methyl-10-ethyl deazaflavine.

[0091] Comparative Example 1

[0092] A method for synthesizing 2-methyl-10-ethyl deazaflavine, the specific steps of which are the same as those of Example 4, except that in step (2), the supernatant containing wild-type imine reductase is used instead of the supernatant containing imine reductase mutant.

[0093] Test Example

[0094] The purity of 2-methyl-10-ethyl apiofungin synthesized in Example 4 and Comparative Example 1 was determined by high performance liquid chromatography, and the method was as follows:

[0095] A Waters Arc 2489 system was used, the chromatographic column was Arlchrom WP C 18 , 4.6*250mm, 5μm; the detector was an ultraviolet detector; the detection wavelength was 270nm; the mobile phase A was 0.01M potassium dihydrogen phosphate solution, the mobile phase B was acetonitrile; the flow rate was 1mL / min; the column temperature was 15℃; the injection volume was 10uL; the gradient operation table was as shown in the following table:

[0096] Time (min) Mobile phase A Mobile phase B 0 90 10 2 80 20 15 80 20 30 50 50 40 50 50 40.1 90 10 50 90 10

[0097] The high performance liquid chromatography detection results of 2-methyl-10-ethyl apiofungin synthesized in Example 4 and Comparative Example 1 are shown in Figure 2 、 Figure 3 .

[0098] As can be seen from Figure 2 、 Figure 3 , the purity of 2-methyl-10-ethyl apiofungin synthesized in Example 4 reached 98.4%, while the purity of 2-methyl-10-ethyl apiofungin synthesized in Comparative Example 1 was only about 76%. This shows that compared with the wild-type imine reductase, the enzyme activity of the imine reductase mutant provided by the application is significantly improved, which can more effectively catalyze the reductive coupling reaction of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione and D-glucose, improves the utilization rate of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione, and greatly improves the synthesis efficiency and purity of 2-methyl-10-ethyl apiofungin.

[0099] Finally, it should be noted that the above only describes the preferred embodiments of the application and is not intended to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. An imine reductase mutant, characterized in that, The amino acid sequence is shown as SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

1.

2. A recombinant vector, characterized in that, is inserting the encoding gene of the imine reductase mutant of claim 1 into a plasmid vector; Preferably, the plasmid vector is pET-28a(+).

3. A recombinant bacterial strain, characterized in that, is transforming the recombinant vector of claim 2 into a host cell to obtain; Preferably, the host cell is Escherichia coli.

4. The imine reductase mutant of claim 1 is used in the synthesis of 2-methyl-10-ethyl deazaflavin.

5. A method of synthesizing 2-methyl-10-ethyldeazaflavin, characterized in that, The steps include: (1) cloning the imine reductase mutant gene of claim 1 into an expression vector to obtain a recombinant expression vector; then transforming the recombinant expression vector into a host cell, selecting a positive clone for fermentation culture, collecting wet bacteria, adding the obtained wet bacteria into PBS buffer, ultrasonic crushing for 35-45 min, then centrifuging at 8000-12000 rpm for 5-15 min to obtain supernatant containing the imine reductase mutant; (2) dissolving (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in dimethyl sulfoxide, stirring until completely dissolved to obtain solution A; adding D-glucose, glutamate dehydrogenase, reduced nicotinamide adenine dinucleotide phosphate into PBS buffer to obtain solution B; (3) adding solution A and the supernatant containing the imine reductase mutant obtained in step (1) into solution B, stirring to mix uniformly, reacting at 35-40℃, 200-300 rpm for 20-30 h, then adding NaOH to terminate the reaction, after extraction and drying, obtaining 2-methyl-10-ethyl deazaflavin.

6. The method of synthesis of claim 1, wherein, In step (1), the PBS buffer has a pH of 7.5, and the mass-volume ratio of the wet bacteria is 10:1, unit: mL / g; the ultrasonic crushing is performed by using an ultrasonic cell crusher, treating at a power of 150-300 kw for 40 min, working for 3 s and stopping for 3 s.

7. The method of synthesis of claim 1, wherein, In step (2), the structural formula of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione is shown as formula (1): It is prepared according to the following method: adding 1-methyl barbituric acid and 2-ethylaminobenzaldehyde into a reaction container containing ethanol to construct a reaction system; stirring the reaction system at 60-90℃ for 4-8 h, after the reaction is completed, cooling the reaction liquid to room temperature, precipitating solids, filtering, and recrystallizing with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione; wherein the mass ratio of 1-methyl barbituric acid to 2-ethylaminobenzaldehyde is 1:(1-1.2); the volume-mass ratio of ethanol to 1-methyl barbituric acid is (10-20):1, unit: mL / g.

8. The method of synthesis of claim 1, wherein, In step (2), the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in the solution A is 0.2-0.3 g / mL.

9. The method of synthesis of claim 1, wherein, In step (2), the concentration of D-glucose in the solution B is 55-65 mM, the concentration of glutamate dehydrogenase (GDH) is 55-65 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 35-45 mM.

10. The method of synthesis of claim 1, wherein, In step (3), the volume ratio of the solution A, the solution B and the supernatant containing the imine reductase mutant is (45-55):(440-460):

10. The termination reaction, extraction and drying are specifically as follows: first, the reaction is terminated by adding NaOH with a concentration of 5 M, then the reaction solution is extracted twice with ethyl acetate with a volume of 3 times of the reaction solution, the organic phases obtained by extraction are combined, and finally the combined organic phase is dried with anhydrous MgSO4.

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